Abstract Small conductance Ca2+-activated K+ (SK) channels are present in sarcolemma (sSK) and inner mitochondria membrane (IMM, mSK) in ventricular cardiomyocytes (VCMs). They have a unique ability to link intracellular [Ca2+] with both plasmamembrane repolarization and mitochondria function. SK channels, although thought to be dormant in health, are implicated in ventricular arrhythmias in animal models and in human patients with heart failure (HF). Heterogeneous upregulation of SK channels can exacerbate substrate for arrhythmia. However, recently accumulated evidence suggests that in HF or Long QT syndrome, SK channels provide protection by mitigating loss of repolarization reserve and by reducing Ca2+-dependent triggers for arrhythmia. The potential of SK channels as a target for anti-arrhythmic therapy is yet to be determined largely because of the lack of understanding of cellular and molecular mechanisms that govern SK function. Our main objective is to unravel mechanisms of regulation of sSK and mSK channels using rat model of hypertrophy and failure induced by thoracic aortic banding (TAB). Our central hypothesis is that both sSKs and mSKs are positively regulated by the serine-threonine kinase PKA and negatively regulated by the Tyrosine kinase Pyk2 via modulating channel responsiveness to Ca2+/voltage-dependent block. We posit that PKA-mediated functional upregulation of sSKs and mSKs plays an adaptive role by attenuating arrhythmic potential in hypertrophic hearts, but that the protection offered is not complete. We therefore reason that further enhancement of sSK and/or mSK activity can be achieved via inhibition of Pyk2-mediated phosphorylation, and that this could serve as a novel approach to decrease arrhythmias in cardiac diseases associated with reduced repolarization reserve and defective Ca2+ homeostasis such as hypertrophy and HF. The Specific Aims are: 1: To determine the mechanisms of functional upregulation of sSKs in VCMs using a TAB rat model of hypertrophy and HF. 2: To determine the mechanisms of mSK upregulation and their role in regulation of RyR2-mediated Ca2+ release in TAB rat VCMs. We hypothesize that mSK upregulation facilitates mitochondria cristae flattening which leads to increase in formation of tertiary supercomplexes (SCs) from elements of Electron Transport Chain (ETC), thereby enhancing ETC efficiency and reducing rate of mito-ROS production resulting in improvement in intracellular Ca2+ homeostasis. At the whole heart level, arrhythmogenic effects of genetic enhancement or inhibition of SK channels will be studied using optical mapping of membrane potential and Ca2+; at the single cell level, myocytes from TAB hearts will be investigated with a combination of patch clamp, confocal microscopic imaging of Ca2+ and ROS using novel subcellular-compartmental biosensors, mitochondrial membrane potential and currents, advanced electron tomography and biochemical approaches. Cardiac-specific delivery with Adeno-associated viral vectors will be used to modify expression levels and targeting of SK channels in TAB hearts.